Correlative Raman Imaging
Raman RISE AFM SNOM•••WITec alpha300 Series
Modular Confocal Raman Microscopy Systems

https://raman.oxinst.com

nucleusLife Sciences

endoplasmic
reticulum

nucleoli

cytoplasm
nuclearmembranemitochondriae
10 µm

Raman image of a cell

vegetative cell
energy storage metabolite
sporesRaman image of Bacillus cereus© A. Hermelink, Robert-Koch-Institute,
Berlin1000 µmgray matter
white matter

Large-area Raman image of the grayand white brain matter of a hamsterbrain

polymer substratedrug-eluting polymer coating
drug

Pharmaceutics &Cosmetics

1000 µm

TrueSurface™ topographic Raman
image of a pharmaceutical tablet

0.5 µmRaman depth-scan image of adrug-eluting stentHigh-resolution AFM phase image of adrug-eluting stent surface showing thepolymer substrate structure with theembedded drug particles

Materials
Science

500 nmHigh-resolution SNOM imageof a latex projection pattern

2 µm

AFM topography image of asteel surface

tensile strain
compressive strain

1 µm

Material stress in silicon imaged
via Raman peak-shift analysis

Geoscience

30 µm

Correlative Raman-SEM (RISE) image of a geological sample with
spectra of its molecular compounds

02

1 µm

AFM image (Pulsed Force Mode)of fossilized bacteria

1 µm

G-layerS2-layer
middle lamella

Raman image of a lignified
plant cell

2 µm

2 µm

Raman-SEM (RISE) image ofa polished cross-section of an
abalone shell

Super-resolution SNOM
image of the nucleus
of a rat hepatocyte

2 µm

AFM image of a cell's
surface

8 µm

Correlative Raman –GFP-fluorescence imageof a eukaryotic cell

Archeology & Arts

900 µm

3D Raman volume image of apharmaceutical emulsion

TrueSurface™ topographic Raman
image of an ancient wall painting

TrueSurface™ topographic Raman imageof paper fibers

3D Raman image of a sheet of paper

1000 µm

10 µm

3D Raman image of a fluid inclusion
in garnet (60 x 60 x 30 μm3)

Correlative Raman-SEM (RISE)
image of hematite and goethite inseveral crystal orientations

Anorthite
Titanite
QuartzFluorite
Organics
CalciteLarge-area Raman image of a polished rock section
with corresponding spectra© C. Heim, Göttingen University

WITec alpha300 Series | 03

Polymer Science,Thin Films &Coatings

AFM

RAMAN

3 µm

10 µm

Correlative Raman (right) – AFM (left) images of a three-component
polymer blend

Raman image of a very thin (7.1 nm)
PMMA film (red) on glass (blue) with
a 4.2 nm alkane contamination layer(green) on top

LowDimensional
Materials

2 µm

Correlative Raman-SEM (RISE) image of a graphene
flake. The colors indicate the graphene layers andwrinklesPeak intensity map of MoSe2 at 19 relative
wavenumbers (cm-1)© M. O'Brien, Trinity College, Dublin

AFM

Photovoltaics &Semiconductors

4 µm

RAMAN

10 µm

Correlative Raman-SEM (RISE)
image of a gallium arsenide sample(yellow: gold substrate; red: GaAs;
blue: residues from production)

4 µm

AFM and Raman images of thesame sample area on a wafer

3D confocal Raman imageof a GaN layer (60 x 15 x 20 μm3)

Food Science

5 µmWhite-light and Raman image overlay of banana pulp

04

6 µm

Emulsion used in food production (redand green: fatty matrix; blue: aqueous
phase; yellow: emulsifier)

AFM

RAMAN-AFM

X-Z RAMAN

10 µm

Raman depth-scan image of theinner multi-layer coating of abeverage container

0.7 µm

3 µm

Correlative Raman-AFM image andRaman x-z cross section (inset) of a
two-component fiber

100 nm10 nm

AFM phase image of long chain
polymer molecules

Correlative Raman-SEM (RISE)
image of a polymer blend

Correlative Raman-SEM (RISE)
image of MoS2 flakes with edges
and defects

3D Raman image of carbon nanotubes(green) on a polymer matrix (red)0.7 µmHigh-resolution SNOM image of a WS2 flake (left) and corresponding
photoluminescence peak position image (right)

Forensics

Nanophotonics

4 µm

Raman image of a fingerprint
with explosive material residues

3D Raman imageof a pollen grain (green) in
crystalline honey (50 x 50 x 50 μm3)

SNOM image of a surface plasmonpolariton wave launched on ananostructured metal grid

TrueSurface™ topographic
Raman image of a dextrose candy

WITec alpha300 Series | 05

alpha300 Series

The Instruments

Ramanalpha300 R

alpha300 RA

alpha300 RS

SNOM
alpha300 A

AFM

alpha300 S

Pioneering techniques, maximum versatility and ease of operation – that is what
WITec, the Raman imaging company, stands for. To meet the most exacting
requirements of our customers, WITec continually develops microscopy systems
for correlative Raman imaging that fulfill three crucial tasks simultaneously:
they provide the best spatial and spectral resolution along with peerlesssensitivity and unmatched speed.

Benefits of the WITec alpha300 R series
Identification and visualization of the distribution of chemical compounds
Analysis of crystallinity and material stress properties• Lateral, diffraction-limited spatial resolution down to ~200 nm• 3D chemical imaging enabled by confocal architecture• Nondestructive• No staining or labelling necessary• Correlative imaging options readily available (i.e. fluorescence microscopy,
AFM, SNOM, SEM, TERS)••

06

Design & Components
Modular & Upgradable

01

02

06

M 07

02

OUTPUT COUPLER*
outputs for additional
spectrometers

03

LASER INPUT COUPLERS*
single & triple wavelength
modules combinable andstackable for more wavelengths

04

VIDEO CAMERA COUPLER*
able to house different
camera models

05

KÖHLER WHITE-LIGHTILLUMINATION*
with adjustable LED intensity

AFM BEAM-DEFLECTION UNIT
for cantilever-based
topography and near-field
optical measurements

03
MICROSCOPE TURRET*
up to 6 objectives

08

STEPPER MOTOR
for z-movement, focusing,
depth profiling, 3D stacks

09

POSITIONING &SCANNING STAGES
cooperative scanning of piezodriven and/or motorized stages

10

01

OUTPUT COUPLER*
primary spectrometer output

04

05

06
MICROSCOPE CONTROL &DATA EVALUATION SOFTWARE
WITec Control
WITec Project
WITec Project Plus
WITec TrueMatch
WITec ParticleScout

07
09

08

*motorized options available

10

WITec alpha300 Series | 07

Confocal Raman Microscopy &3D Raman Imaging
The Raman principle
The Raman effect is based on inelastic scattering of
excitation light by the molecules of gaseous, liquid or solidmaterials. The interaction of a molecule with photons
causes vibrations of its chemical bonds, leading to specificenergy shifts in the scattered light that can be identified in
its Raman spectrum.
• Any given chemical compound can be easily identified bythis individual spectral “fingerprint”.
•

a

b

c

d

Additional sample information from Raman spectra:
a. Peak intensity: Quantity of a specific compoundb. Peak shift: Identification of stress and strain statesc. Peak width: Degree of crystallinity
d. Polarization state: Crystal symmetry and orientation

Raman imaging
When Raman spectra are collected at every
measurement point using a confocal microscope
combined with a spectrometer, a Raman image canbe generated that visualizes the distribution of thesample's compounds. Due to the high confocality of
WITec Raman systems, volume scans and 3D images
can also be generated.

08

Resolution

simultaneously
Resolution: Lateral resolution is physically limited
to ~200 nm, depending on the wavelength of theincident light.

Speed

Speed: The more sensitive a system is, the shorter
the acquisition time for a single spectrum. WITec's
Ultrafast Raman Imaging reduces acquisition times
for single Raman spectra down to well
below 1 ms.
Sensitivity: A high confocality increases thesignal-to-noise ratio by reducing the background.
With the UHTS series, WITec developed lensbased, wavelength-optimized spectrometers with
an ultimate spectral resolution down to 0.1 cm-1
relative wavenumbers (@633 nm excitation).

Sensitivity

No need for compromises
The Raman effect is extremely weak, so every
Raman photon is important for imaging.
Therefore WITec Raman imaging systems
combine an exceptionally sensitive confocal
microscope with an ultrahigh-throughput
spectrometer system (UHTS).
The precise adjustment of all optical andmechanical elements guarantees the highest
resolution, outstanding speed andextraordinary sensitivity simultaneously!
This optimization allows the detection ofRaman signals of even weak Raman
scatterers and extremely low material
concentrations or volumes with the lowest
excitation energy levels. This is an unrivaled
advantage of WITec systems.
3D Raman image of a honey droplet
containing a pollen grain (green) andseveral crystalline phases (red, blue,cyan), reconstructed from 50 individual
2D Raman images acquired along thez-axis. Scan range: 50 x 50 x 50 μm3

WITec alpha300 Series | 09alpha300 R

Raman Microscope

The alpha300 R is a confocal Raman microscopy system available inseveral configurations with different features to fulfill a wide variety ofcustomer requirements.
Whether you want to perform micro-Raman mapping or high-end
3D Raman imaging, as a Raman newcomer or an experienced user,
in a scientific setting or an industrial laboratory, WITec offers the perfect
Raman imaging solution for you.

Key features and benefits
True confocality, ideally suited to depth profiling and 3D Raman
image generation• Lateral resolution limited only by physical law• Spectral resolution down to 0.1 cm-1 relative wavenumbers(@633 nm excitation)• Focus stabilization compensates for thermal and mechanicalvariations during long-term measurements• Laser wavelength selectable from UV to NIR• Throughput-optimized UHTS spectrometers with a variety of focal
lengths• Fast Raman Imaging™ and Ultrafast Raman Imaging withmotorized or piezo-driven scanning stages• 3D images and depth profiles with motorized or piezo-driven
scanning stages•

10alpha300 access
The alpha300 access is a high-quality confocal micro-Ramansystem that provides an entry point to WITec's technology.

Key features and benefits
Class-leading capability within challenging budget andprocurement environments• High-performance Raman mapping• Exceptional spectral quality provided by the WITec
UHTS Raman spectrometer series• High-quality and ultra-precise optical components• WITec Raman and imaging know-how• The future of Raman spectroscopy
through upgradeability•

Raman lab arrangement

UHTS spectrometer
Microscope

Remote control unit
WITec software suite

alphaControl

Laser
WITec alpha300 Series | 11

alpha300 apyron™

Raman Microscope

As a member of the WITec microscope family, the alpha300 apyron™
sets the benchmark for automated Raman imaging systems withexcellent imaging quality, outstanding spectral and spatial resolution,
ultrafast acquisition times and exceptional signal sensitivity. It is ahighly-automated, push-button instrument. An extensive set ofdefault and advanced measurement parameters in combination withmotorization and a powerful software environment allow the user toobtain optimal results with unprecedented ease.

High spatial and spectral resolution simultaneously: CCl4 in an emulsion
B

CD

Intensity (a.u.)A(A) 3D confocal Raman image of an emulsion with corresponding Raman
spectra shown in (C) green: alkane; blue: water; yellow: carbon tetrachloride(CCl4) + oil. Image parameters: 200 x 200 x 20 pixels, 100 x 100 x 10 µm3
scan range, 0.06 s integration time per spectrum, 532 nm excitation
wavelength. (B) Zoom-in image with high spectral resolution. (D) Due tothe high spectral resolution of the spectroscopic system, the CCI4 band at
460 cm-1 relative wavenumbers can be resolved at room temperature
into three peaks that reflect the isotopic variations of the molecules.

12alpha300 Ri

Inverted Confocal Raman Microscope

The alpha300 Ri turns chemical imaging upside down.
Its inverted beam path preserves all the functionality of
WITec's alpha300 R microscopes while introducing
a new angle in access and handling. The ability toview and investigate samples from below is a great
advantage when working with aqueous solutions oroversized samples. Studies in life sciences, biomedicine,
pharmaceutics and geosciences in particular will benefit
from the consistency and flexibility provided by thegeometry of the alpha300 Ri. Many accessories andupgrade possibilities developed for the alpha300 series
are compatible with the Ri version.

Bulky samples that would be challenging toinvestigate underneath a conventional microscope
objective turret can be accommodated by placing
them on the stage of the alpha300 Ri.

An inverted beam path allows liquid samples
to be placed on the fixed plane of the stage
for quick and repeatable measurements.

The motorized sample stage
also facilitates the mounting ofenvironmental enclosures andother accessories.

WITec alpha300 Series | 13

alpha300 A

Atomic Force Microscope

The WITec alpha300 A atomic force microscope integrated with a research-grade
optical microscope provides superior optical access, easy cantilever alignmentand high-resolution sample survey.
Using optical pre-inspection by means of various illumination and detection
techniques (e.g. bright field, dark field, polarization, fluorescence, etc.), theuser can conveniently determine the area of interest for the AFM measurement.
By simply rotating the microscope turret, the user can switch between
conventional microscopy and AFM modes quickly and easily.
The WITec AFM objective provides a direct view of both sample and cantilever
for straightforward and precise cantilever tip positioning. The alpha300 A system
includes an extremely linear and precise capacitive feedback-controlled scanningstage featuring TrueScan™ for exceptional accuracy over the entire scan range.

Key features and benefits

AFM modes

Surface characterization on the nanometer scale
Optical and AFM combination• Nondestructive imaging• Minimal, if any, sample preparation• Ease of use in air and liquids• Combinable with confocal Raman imaging and scanningnear-field optical microscopy (SNOM)• TrueScan™ controlled piezo-driven scanning stages withcapacitive feedback loops:– 30 x 30 x 10 μm3– 100 x 100 x 20 μm3– 200 x 200 x 20 μm3

AC ModeContact Mode
• Digital Pulsed Force Mode™ (DPFM)• Electrostatic Force Microscopy (EFM)• Kelvin Probe Microscopy• Lateral Force Microscopy (LFM)• Lift Mode™
• Magnetic Force Microscopy (MFM)• Nanomanipulation/Lithography

•

•

•

•

Simultaneous cantilever and sample view for easy determinationof the measurement position
AB

219 nm

C

0 nm

14

The WITec AFM objective allows simultaneous sample and cantilever survey fromabove with high resolution. It facilitates accurate and convenient AFM tip alignment
and positioning for even very small sample structures. (A) Simultaneous cantileverand sample view, (B) overlay of the optical image with the AFM image (AC mode,
Scan range: 3 x 3 μm2), (C) 3D representation of the AFM image from (B).

Imaging of surface properties with DPFM
A

B
Free Cantilever
Oscillation

BaselineyyForce Signal

Force

Fmax
Snap

Adhesionyx
Stiffness
Adhesionx
Stiffness

Contact
Time
Viscosity

Contact Time

Topography

Topographyx
Stiffness

Viscosity

Contact Time
Viscosity

Adhesion
Peak

Time

Digital Pulsed Force Mode (DPFM)
Pulsed Force Mode (PFM) is a non-resonant, intermittent
contact mode for atomic force microscopy that allows
the characterization of material properties such as
adhesion, stiffness and viscosity along with the sample
topography. Additionally, lateral forces are virtuallyeliminated. Therefore high-resolution mapping of delicate
samples in air and liquids is easily achievable while
maintaining a scanning speed comparable to contact
mode AFM. In contrast to most other intermittent contact
techniques, the perpendicular forces on the sample(introduced by the AFM tip) are controlled by a feedback
loop.

Topography(A) The PFM electronics induces a sinusoidal modulation of the z-piezoof the AFM with an amplitude of 10 – 500 nm at a user-selectablefrequency of between 100 Hz and 2 kHz, which is far below theresonant frequency of the cantilever. A complete force-distance cycle
is carried out at this rate, resulting in the force signal as shown in the
figure. (B) An ethyl-hexyl-acrylate/polystyrene blend (EHA/PS) was
spin-coated onto a glass substrate. Various surface properties wereanalyzed and imaged simultaneously using DPFM. Dark areas in theimages correspond to low values.

WITec alpha300 Series | 15alpha300 S

Scanning Near-field Optical Microscope

Super-resolution microscopy down to 60 nm can beachieved with the WITec scanning near-field optical
microscope (SNOM) alpha300 S. SNOM, as compared toother optical super-resolution techniques such as STEDor STORM is not dependent on fluorescent tags or
specialized laser excitation sources. The operation of thesystem is straightforward as various measurement
procedures such as a high-speed automatic cantileverapproach and adjustment can be controlled through theintuitive software.

A

B

The alpha300 S combines the advantages of SNOM,
confocal microscopy and AFM in a single instrument.
By rotating the objective turret one can easily switch
between the different modes. One field that can benefit
immensely from this technique is nanotechnology
research, in particular nano-photonics and nano-optics.
In life sciences and materials research, SNOM permits
optical detection of the most miniscule surface structuresof transparent as well as opaque samples.

C(A) Video camera top view of a SNOM sensor and sample, (B) side viewof a cantilever pyramid, (C) SNOM cantilever wafer.
Comparison of optical resolution between confocal microscopy (D) andscanning near-field optical microscopy (E) on a latex projection pattern.

Cantilever SNOM-sensors
The alpha300 S uses unique, patented, high-quality microfabricated SNOM sensors consisting of a silicon cantileverwith a hollow pyramid tip. The SNOM aperture is at the apexof the pyramid. The laser light used for optical imaging isfocused into the back of the tip and then onto the sample.
Due to the wide opening angle of the pyramid, thetransmission coefficient is much higher than that of fiber
probes with the same aperture diameter. An established and16

proven manufacturing process produces tips with aperturesof varying sizes to meet customers' individual requirements.
Cantilever SNOM sensors are, unlike fiber tips, very robust andflexible in the z-direction and allow the beam deflection
technique to precisely control the tip-sample distance. All ofthese innovative characteristics make the handling of probes
during near-field measurements very user-friendly for themost reliable optical imaging available beyond the diffraction
limit.

Key features and benefits
Spatial resolution beyond the diffraction limit(ca. 60 nm laterally)• Unique patented SNOM sensors• Ease of use in air and liquids• Nondestructive, label-free imaging techniqueprovides super-resolution microscopy withminimal, if any, sample preparation• Upgradeable with confocal Raman imaging
for correlative microscopy and near-field
Raman imaging• Three techniques always integrated within one
instrument: confocal microscopy, AFM and SNOM•Super-resolution microscopy: SNOMD

1 µm

Confocal microscopy,
ca. 300 nm resolution

E

1 µm

SNOM, ca. 60 nm resolution

WITec alpha300 Series | 17

Correlative Raman Imaging					Made Easy
WITec's alpha300 microscope series was designed with
inherent flexibility and configurability to address users'
individual requirements. This not only involves easy andcost-effective upgrade and extension options, but also
the possibility to add other microscopy techniques to the
instrument.
As no single imaging approach can reveal every featureof a sample, a more comprehensive understanding canbe achieved by using several techniques and correlating
the results.

This advantage is particularly pronounced when using
methods that deliver images of the morphology of asample while also determining its molecular composition.
WITec's modular product line incorporates nearly all
scanning probe and optical microscopy techniques. Eachmicroscope model can always be equipped with thefunctionality of another variation of the alpha300 family
either as a built-in feature or as a later upgrade. The WITec
hardware and software environment is used for all newfeatures and upgrades, ensuring the best possible
compatibility and ease of use.

Switching microscopy modes by rotating the objective turret
Scanning Probe Microscopy (SPM) mode for AFM
or SNOM: The SPM cantilever is held at the end of
the objective's arm. The cantilever is aligned with an
integrated highly-precise inertial drive.

18

Optical modes: Rotating the turret provides access to theoptical modes, allowing high-resolution confocal opticaland chemical imaging to be seamlessly linked with AFM
or SNOM measurements without transferring the sample.

A

B

Raman-AFM
By combining the materials analysis capability of confocal
Raman imaging with the ultrahigh topographic and lateral
resolution of an AFM, the chemical properties of a sample
can be easily correlated with its surface characteristics.
The well-established alpha300 RA was the first integrated
Raman-AFM system on the market and it continues to set
the benchmark for combined instrument configurations.

C

Correlative, high-resolution AFM-confocal
Raman imaging of a polymer mixture(A) The sample's phase image was created using AFM in tapping
mode. (B) Raman spectra were recorded at each image pixel of thearea and displayed as a false color image. Red: polystyrene, green:
ethyl-hexyl-acrylate (EHA), violet: styrene-butadiene-rubber (SBR),
blue: mixed spectrum of SBR and EHA. (C) The overlay of the AFM
phase and the Raman image identifies and locates the sample's
components.

The alpha300 RA incorporates the features of thealpha300 R for chemical imaging along with thealpha300 A for atomic force microscopy.
For TERS options, please see page 23.

Raman-SNOM
The alpha300 RS combines Raman characterizationand imaging with scanning near-field optical
microscopy for optical imaging with resolution beyond
the diffraction limit. It combines all features of thealpha300 S and alpha300 R instruments.

alpha300 R and alpha300 S

A

B

C

1 µm

(A) Near-field Raman image of the G-Band intensity of graphene. (B) The graph shows
the G-band intensity along the red line and reveals the measurable signal variations
between the small sample and the substrate. (C) Corresponding Raman spectrum
obtained by the Raman-SNOM measurement.

WITec alpha300 Series | 19

RISE™ – Raman Imaging

and Scanning Electron Microscopy

Confocal Raman imaging and scanning electron
microscopy (SEM) are techniques that are ideally suited tocorrelating structural and chemical information.

Principle of RISE™ microscopy
Samples are automatically transferred from one measuring
position to the other within the vacuum chamber of the
combined Raman-SEM instrument, streamlining theworkflow and drastically improving ease of use.

20

Key features and benefits
RISE™ microscopy systems combine all features of a standalone SEM and a WITec research-grade confocal Raman
imaging microscope within one instrument to provide:• Quick and convenient switching between Raman and
SEM modes• Automated sample transfer from one measuring position
to the other within the vacuum chamber• An integrated software interface for user-friendly
measurement control• Easy correlation of experimental results and image overlay• SEM and Raman imaging capabilities without compromise• A truly confocal optical path
• Research-grade optical imaging

The sample remains inside the vacuum chamber during both theSEM and Raman measurements to ensure seamless correlative
microscopy.

Application iron mineralogy
B

C

Intensity (a.u.)

A

1o µm

(A) In the SEM image, structural characteristics of a piece of hematite (Fe2O3) are visible. (B) Hematite, goethite and vonsenite wereidentified by their Raman spectra. The spectra of crystal forms of hematite are depicted in green, orange and purple, those of goethite(FeO(OH)) in red and cyan. The spectrum of vonsenite is shown in blue. (C) Correlation of Raman and SEM data produced a RISE image.

WITec alpha300 Series | 21

Fiber-Based Beam Delivery

Facts and Benefits

WITec's modular design relies on efficient beam delivery via optical fiber. Long-term investments inresearch and development have yielded an exceptional understanding of fiber-coupling mechanisms,
enabling benefits unattainable with more conventional methods.
Virtually lossless energy transmission
Fibers provide a diffraction-limited point light source for the highest confocality and spatial resolution• Pre-configured and pre-aligned fiber coupling units guarantee long-term stability and user-friendlinesswithout the need for further adjustment• Polarization direction of the light is maintained for the most intricate polarization-dependentmeasurements• Lasers and spectrometers can be mounted far away from the microscope, allowing flexible andcompact system footprints that can alleviate thermal or vibrational disturbances••

22Modular Components & Accessories

Beam Path Options

Excitation and detection options

alpha300 R

The WITec alpha300 microscope series features several
beam path geometries. The upright microscope enables
measurements of opaque samples while the inverted
microscope (p. 13) is ideal for experiments on transparent
samples. Side-illumination options are also available.
The modularity and inherent flexibility of WITec's uniquealpha300 microscope systems provide optimized
instrumentation for many different types of experiments.

Excitation & detection
from above

alpha300 Ri
Excitation & detection
from below

TERS working principle
WITec TERS module for the alpha300 series
Tip-Enhanced Raman Spectroscopy (TERS) enables
the acquisition of chemical information with a lateral
resolution far below the diffraction limit. The TERS tip
illumination can be performed from either above, below
or from the side. The WITec alpha300 microscope
series accomodates all excitation approaches and TERS
measurements can be carried out alongside Raman
and AFM investigations with the same integrated
instrument.

AFM cantileverAg- or Au-coated
AFM tip

Diffraction limited
focus spot

Strong localized
field enhancement

Laser

alpha300 TERS experiment on a carbon nanowire: The
enhancement of the spectral intensity in TERS-mode isclearly visible.
WITec alpha300 Series | 23

Electronics & Software
alphaControl
alphaControl is a patented digital microscope controller basedon the system-on-a-chip concept. The digital component of
the system is entirely realized within one Field Programmable
Gate Array (FPGA). This architecture results in speed, flexibility,
accuracy, expandability and precise timing, while simplifying
operation of the various microscope configurations andextensions from a single hardware device.

Key features and benefits
TruePower™ for precise and absolute laser
power adjustment for up to four lasers• Automated high-speed cantilever approach• High-speed frequency scan
• TrueScan™ lag error correction• Automated light intensity adjustment• High-speed overload protection forphoton-counting detectors• Two built-in fully digital lock-in amplifiers(real amplitude and real phase up to 500 KHz)
• Fully digital feedback-control• USB 3.0 interface• Digital piezo controller interface• Real-time rotation matrix and real-timetranslation for XYZ scans• Precise timing• Modular and upgradable system

TrueScan™

•

1 µm

1 µm

TrueScan™ is an integrated scanning engine that corrects
dynamic positioning errors. As the piezo-driven scanningstage moves the sample forward and backward during
the measurement the stage's inertia leads to lag errors.
This effect increases with scanning speed. While theincorporated capacitive feedback system determines theactual position of the stage, TrueScan™ can resolve thediscrepancy between its actual and intended position,
allowing high-resolution lag-free AFM imaging at highscanning speeds.

24

WITec Suite SIX

Powerful Software Environment

Experimental setup, instrument control and data analysis are made easy with WITec's
software tools.
Sophisticated data acquisition, evaluation, post-processing and image generation
for confocal Raman microscopy, AFM and SNOM.
• The software architecture and graphical user interface offer an integrated andconsolidated functionality incorporating every technique and measurement mode.
• Suitable for all experience levels and user requirements through an individuallyadjustable user interface.
•Control SIX

Project SIX

Project SIX+

WITec Project SIX

WITec Project SIX+

WITec microscope family with alphaControl

WITec Control SIX
Centralized experiment and instrument settings• Acquisition of imaging and/or spectroscopy data
• On- and offline data analysis and image processing•

Offline data and image
processing• Licensed for an unlimited
number of users•

Advanced offline data and image
processing• WITec Project functionality plus
additional features for advanced
data analyses• Single user license•

WITec alpha300 Series | 25

Modular Components & Accessories
01

UHTS 300 VIS

02UHTS 600 VIS

03 UHTS 400 UV - VIS
04

BROADBAND UHTS 300 VIS - NIR

05

UHTS 400 NIR

06
400 nmUV

500 nm

600 nm

700 nmVIS

UHTS 300 IR

900 nm1100 nm1300 nm

1500 nm1700 nm

NIR

Spectroscopy solutions: The UHTS series
The WITec Ultra-High Throughput Spectrometer (UHTS)
series consists of exceptionally flexible and precise devices
that meet the demands of cutting-edge confocal Raman
imaging.
Varied excitation sources and scattering experiments
require specialized optics for optimal throughput. The
WITec UHTS design approach takes this into account by
employing a series of lens-based on-axis spectrometers
for a range of excitation wavelengths.

Sample positioning and scanning stages

Heating & cooling stages

WITec offers manual, stepper motor-driven and piezoactuated stages to accommodate a wide variety ofsamples and applications.

For experiments requiring an accurate temperature control,
the alpha300 series can be equipped with heating andcooling stages with ranges from -269°C up to 1500°C (4 K 1773 K) for AFM and high-resolution Raman measurements.

Manual translation stage for accurate sample positioning
Motorized translation stages for large-scale positioning,
mapping and imaging• Piezo-driven scanning stages with closed-loop operation
for AFM and high-resolution Raman measurements••

The stages feature several different travel ranges for thehighest flexibility.

26

Spectrometers of the UHTS series allow more than 70%
total transmission for high-speed and high-resolution
Raman imaging. They feature an optical fiber port anda motorized triple-grating turret. Customers can choose
from among several focal lengths and gratings to match
individual requirements in terms of spectral range orresolution.

Sample mounting stages
A selection of mounting stages is available for securing
samples of various shapes and environmental
requirements. Microscope slides, Petri dishes, NUNC™
flasks and cylindrical samples such as stents can all beaccommodated. The integration of third-party cryostats isavailable upon request.

TrueSurface™

1000 µm

TrueSurface™ microscopy

Laser sources

WITec's patented TrueSurface™ microscopy option enables
confocal Raman imaging guided by surface topography.
With TrueSurface™ chemical characterization on rough,inclined or irregularly-shaped samples can be carriedout precisely because the sensor actively monitors the
sample surface and keeps it in focus. This topographic
imaging module also compensates for variations during
measurements with long integration times. TrueSurface™
pioneered by WITec uses an advanced optical profilometer
integrated within the instrument to provide one-passsimultaneous acquisition of both topographic andmolecular information.

Excitation laser sources for Raman, SNOM andphotoluminescence imaging can be individually chosen
and attached in concert to the microscope via opticallymatched fiber coupling. Available laser wavelengths range
from the UV to the NIR to fulfill a variety of experimental
excitation requirements. Switching between different lasers
is a matter of simply rotating a wheel in the coupling unit orby a click in the software.

Laser power adjustment with TruePower™

0

TruePower is a new experience in laser power adjustment
for confocal Raman imaging measurements. The absolute
laser power delivered to the microscope is measured andadjusted with an accuracy of 0.1 mW. A laser shutter shields
the sample from the laser light and opens only during
Raman analysis with an optimized power level to avoid
any sample degradation. TruePower is the only laser power
regulation system on the market to provide such accuracy.

mW
3

3.5

4

4.5

5

100

Optimal laser power determination for the preservation ofdelicate samples and reproducibility in measurement 		
conditions• Power series measurements enabled by making powerinduced spectral changes quantifiable• Automated documentation of laser settings• Retrofittable for most WITec imaging systems•

WITec alpha300 Series | 27

Modular Components & Accessories- Simultaneous multi-spectra
search (1:1 match)- Multiple component search:combine up to threedatabase components todescribe a measured
composite spectrum

Control

Spectral data acquisition

500

TrueMatch

400
300

CCD cts

100

CCD cts

200

400

Spectral data evaluation& post-processing

500

Project- Automated and simultaneous
demixing analysis of multiple
components

500

1000

2500
2000
1500
relative wavenumbers (cm-1)

2500
1500
2000
relative wavenumbers (cm-1)

3000

3000

3500

3500

TrueMatch™ Raman spectral databasemanagement

RayShield coupling unit for Raman imaging at
low wavenumbers

TrueMatch™ is a powerful and innovative software module
for identifying the components of a sample and foraccessing and developing Raman spectral databases.
With this technology, existing databases containing
representative Raman spectra of common materials or
substrates can be searched and referenced. It also allows
users to create their own catalog of spectra pertinent
to their research. TrueMatch™ is fully integrated with the
WITec Suite SIX software environment and is a perfectcomplement to its data evaluation andpost-processing capabilities.

The RayShield coupler includes a specialized narrow band
filter set which is optimally aligned for the detection of
Stokes and anti-Stokes Raman lines close to the Rayleighline (ca. 10 cm-1) while maintaining an ideal Rayleighshielding.

ParticleScout
ParticleScout is a Raman imaging tool that can find, classify,
identify and quantify particles in a sample. It enables
measurements that move from an overview survey throughtargeted investigations of particles grouped by attributes tothe precise chemical characterization of individual particles.
ParticleScout leverages the speed, sensitivity and resolutionof WITec’s Raman imaging microscopes to ensure quick andseamless particle analysis.

28

1000

Anti-Stokes extension
A wavelength-optimized filter unit for the detection ofanti-Stokes Raman signals is available as an option foradvanced Raman imaging experiments.

Polarization module
Polarization dependent measurements can be used
to analyze molecular orientations and geometries ofmaterials. The module is comprized of an excitation
wavelength specific polarization kit for rotating thepolarization of the laser light and an analyzer for filtering
information of a certain polarization.

StrobeLock
StrobeLock is a WITec extension that enables timecorrelated single photon counting measurements ofunprecedented accuracy. The available imaging modes
include fluorescence lifetime imaging microscopy (FLIM).

Correlative Raman – fluorescence microscopy image of eukaryotic
cells. Nuclei were stained with DAPI (blue). Endoplasmic reticulum (red)and nucleoli (green) were identified by their Raman signals.

nucleoli
DAPI

ER

Enclosure & rigid support frame

AFM & SNOM cantilevers

For maximum reduction of environmental interference suchas acoustic or vibrational noise, the WITec support frameand enclosure system greatly contributes to achieving
exceptional imaging results. The enclosure can be
configured as an air-tight option to allow control of thegas-phase during experiments. Equipped with an interlock,safety regulations (e.g. laser class I requirements) can beeasily accommodated.

WITec provides several types of high-quality AFMcantilevers for various AFM imaging modes. WITec is the
only company capable of producing highly reliable SNOM
cantilever sensors with apertures ranging from 60 nm to
100 nm. Each SNOM cantilever is individually tested before
shipment.

Objectives
A wide range of objectives for various microscopy
techniques are available to meet virtually any
experimental requirement.

Imaging in liquids
Imaging in liquids can be easily performed by using
a water immersion objective in optical mode or theliquid imaging extension for AFM and SNOM modes.
A specialized cantilever holder and inertial drive allows
operation in fluids (e.g. in Petri dishes).

LabVIEW interface
The optional LabVIEW interface provides access foralphaControl in order to design and control individual
measurement procedures with LabVIEW.

Customized solutions
If your application requires other components, WITec
looks forward to discussing your individual requirements
and helping to get your experiment up and running. The
inherent modularity of our product line allows additional
high-quality parts and detectors to be easily integrated.

AC (Tapping™) mode for AFM & SNOM
AC mode for WITec microscope systems allows stateof-the-art resonant intermittent-contact AFM and SNOM
imaging specifically tailored for soft and delicate samples.
Acoustic AC mode enhances the imaging capabilities ofcontact mode AFM on such samples, eliminating lateralforces and delivering optimized resolution.

DaVinci nanolithography
Advanced nanotechnology often requires accurate
and reliable nano-manipulation or nanolithography
tools for precise surface structuring. The WITec DaVinci
nanolithography package allows these kinds ofexperiments in AFM as well as in optical modes with an
integrated laser shutter control.

Featured light microscopy techniques
The alpha300 series offers a selection of sample survey
and data acquisition methods such as: fluorescence
contrast imaging, DIC, dark field microscopy, petrography
and polarization-dependent measurements.

Photoluminescence detection
For the detection of photoluminescence spectra beyond
1.1 μm, an InGaAs detector can be easily integrated withthe spectroscopic setup. Raman experiments can beextended to acquire additional information in the nearinfrared photoluminescence regime (or for 1064 nm Raman
measurements).

WITec alpha300 Series | 29

WITecThe Company
Since its founding in 1997, the Raman imaging company
WITec has established itself as a market leader in the fieldsof Raman microscopy and correlative Raman-AFM,
Raman-SNOM and Raman-SEM (RISE) microscopy. WITec's
innovative spirit has kept the alpha300 microscope series
at the forefront of the Raman imaging market since itinitially revolutionized the field and established Fast Raman
Imaging™ as a standard technique. Ongoing developmentof the first truly confocal Raman imaging system continues
to enable the setting of benchmarks in sensitivity, speed
and spectral and spatial resolution. As reflected in WITec's
maxim “Focus Innovations” our success is based oncontinually introducing new technologies and acommitment to maintaining customer satisfaction throughhigh-quality, flexible and innovative products.

Core Competencies
•

•

•

•

•

In September 2021, WITec became a member of the Oxford
Instruments Group, bringing unmatched expertise in Raman
microscopy to its extensive portfolio of businesses.

All WITec products are developed and produced at WITec
headquarters in Ulm, Germany, which accelerates
advancement and ensures the most stringent quality control.
The modularity of our product line delivers unparalleledflexibility, upgradeability, and opportunities for creating
specialized customer solutions.
Our extensive sales and support infrastructure provides
responsive, precise consultation and service in every global
region.
WITec microscopes can integrate several analytical methods
for correlative investigations and offer industry-leading
speed, sensitivity and resolution – simultaneously.
Our instruments are renowned for their reliability and userfriendliness, which allows our customers to focus on thenovel scientific challenges of their experiments.

Achievements
Developments:

Awards:1997 – WITec is founded, alphaSNOM is introduced: the firstcommercial cantilever-based scanning near-field optical
microscope, Pulsed Force Mode intermittent contact method
is developed

2008 R&D 100 Award

1999 – alpha300, the first production confocal Raman
imaging microscope pioneers Raman spectroscopy as a toolfor 3D chemical characterization2012 Photonics Prism Award

2003 – alpha300 RA, the first integrated Raman/AFM		
microscope establishes correlative Raman microscopy as ananalytical technique
2010 – TrueSurface originates topographic Raman imaging
microscopy using an integrated optical profilometer2014 – RISE (Raman Imaging and Scanning Electron)
microscopy enables chemical and structural characterization
within a common vacuum chamber
2015 – alpha300 apyron microscope fully automates
confocal Raman imaging
2019 – ParticleScout automated microparticle analysis toolfinds, classifies and identifies particles using an integrated
Raman database2021 – cryoRaman, cryogenic Raman microscope developedwith attocube systems AG, provides chemically sensitive
imaging at 1.8K
30

2011 PITTCON Editors Gold, R&D 100 Award,
Microscopy Today Innovation Award

2015 Photonics Prism Award,
Achema Innovation Award
2019 SelectScience Scientists’ Choice Award,
The Analytical Scientist Innovation Award,
Wiley Analytical Science Award
2020 The Analytical Scientist Innovation Award
2021 Microscopy Today Innovation Award,
Wiley Analytical Scientist Award,
The Analytical Scientist Innovation Award
2022 R&D 100 Award

WITec Headquarters
WITec Wissenschaftliche Instrumente und Technologie GmbH
Lise-Meitner-Str. 6, D-89081 Ulm, Germany
Phone +49 (0) 731 140 700, Fax +49 (0) 731 14070 200
info@witec.de, https://raman.oxinst.com

WITec alpha300 Series | 31

WITec Product Line
The WITec product portfolio includes imaging
systems for Raman, AFM and SNOM analyses assingle technique solutions as well as correlative
imaging configurations (Raman-AFM, RamanSNOM, Raman-SEM). All WITec microscopes are
high-quality modular systems with exceptional
optical throughput, unparalleled signal sensitivity
and outstanding imaging capabilities.

alpha300 R

The common thread throughout is that all systems
are based on the same hardware and softwarearchitecture. Whenever required it is possible tosimply upgrade any system, even the most basic,
with additional features and components, allowing
our customers to keep pace with future challenges.

The WITec alpha300 R is a research-grade microscope combinedwith an ultrahigh-throughput spectrometer (UHTS) for Raman
imaging with exceptional performance in speed, sensitivity,
confocality and optical and spectral resolution.

32

alpha300 A

alpha300 S

The WITec alpha300 A atomic force microscope provides nanoscalesurface characterization with an integrated research-grade optical
microscope for easy cantilever alignment and high-resolution
sample survey.

The alpha300 S is a super-resolution scanning near-field optical
microscope (SNOM) that combines the advantages of SNOM,
confocal microscopy and AFM in a single instrument. It uses uniquemicro-fabricated SNOM cantilever sensors for spatial resolution wellbeyond the diffraction limit.

alpha300 apyron™
The WITec alpha300 apyron™ is an intuitive motorized
microscope system for Raman spectroscopy andchemical imaging. The push-button principle of thesystem has the advantage of drastically reducing thetime required to become familiar with the operation of
the instrument, which greatly accelerates the initiationof measurements and increases the rate of sample
turnover. With the alpha300 apyron‘s straightforward
interface the user can concentrate solely on theirexperiment.

alpha300 RA

alpha300 RS

The alpha300 RA incorporates the features of the alpha300 R
along with those of the alpha300 A. This instrument enables
correlative Raman-AFM imaging for a more comprehensive
understanding of samples.

The alpha300 RS facilitates confocal Raman imaging in
combination with scanning near-field optical microscopy
for optical imaging with super-resolution down to 60 nm.
This microscope combines all features of SNOM andconfocal Raman imaging and many AFM modes.

WITec alpha300 Series | 33

WITec Product Linealpha300 Ri
The inverted beam path of the alpha300 Ri preserves all thefunctionality of the standard alpha300 R microscope while
introducing a new angle in access and handling. The ability toview and investigate samples from below is a great advantage
when working with aqueous solutions and oversized samples.

34

alpha300 access

RISE™ Microscopy

The alpha300 access is a micro-Raman single-spot analysis
and mapping microscope. It was specifically engineered forbudget-conscious customers with high demands on instrument
performance. It offers outstanding spectral quality, opticalthroughput and signal sensitivity. It is fully compatible with andcan be upgraded by adding alpha300 R series components.

RISE™ microscopy is a novel correlative microscopy techniquethat integrates SEM and confocal Raman imaging. With RISE™
microscopy ultra-structural surface properties can be linked tomolecular compound information. The instruments combine allfeatures of an alpha300 R confocal Raman imaging microscopewith a stand-alone SEM.

WITec alpha300 Series | 35

alpha300 S:
Scanning Near-field
Optical Microscopealpha300 A:
Atomic ForceMicroscopealpha300 R:
Confocal Raman
Microscopealpha300 apyron™: AutomatedConfocal Raman Microscope

WE TAKE CARE – WITec uses environmentally friendly printed materials.
While this policy is only a small contribution to a healthy environment, we at
WITec believe that focusing on details can effect positive change in the world.

WITec Microscopesalpha300 Ri:
Inverted Confocal
Raman Microscope

RISE®: Raman Imaging
and Scanning Electron
Microscopealpha300 access:Confocal Micro-Raman System

Find your regional WITec and Oxford Instruments
contact at https://raman.oxinst.com/contact
WITec Headquarters

Explore our product line:

WITec Wissenschaftliche Instrumente und Technologie GmbH
Lise-Meitner-Str. 6, D-89081 Ulm, Germany
Phone +49 (0) 731 140 700, Fax +49 (0) 731 14070 200
info@witec.de, https://raman.oxinst.com

https://raman.oxinst.com/products/